Immunoconjugates Targeting HER2

ABSTRACT

The invention provides an immunoconjugate of formula:or pharmaceutically acceptable salt thereof, wherein subscript r is an integer from 1 to 10, subscript n is an integer from about 2 to about 25, and “Ab” is an antibody construct that has an antigen binding domain that binds HER2. The invention further provides compositions comprising and methods of treating cancer with the immunoconjugate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 17/465,284, filed Sep. 2, 2021, which is a continuation ofInternational Patent Application PCT/US2020/022645, filed Mar. 13, 2020,which claims the benefit of U.S. Provisional Patent Application No.62/819,356, filed Mar. 15, 2019, each of which is incorporated byreference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 28,689 Byte ASCII (Text) file named“763418_SequenceListing.txt,” created Jun. 23, 2022.

BACKGROUND OF THE INVENTION

It is now well appreciated that tumor growth necessitates theacquisition of mutations that facilitate immune evasion. Even so,tumorigenesis results in the accumulation of mutated antigens, orneoantigens, that are readily recognized by the host immune systemfollowing ex vivo stimulation. Why and how the immune system fails torecognize neoantigens are beginning to be elucidated. Groundbreakingstudies by Carmi et al. (Nature, 521: 99-104 (2015)) have indicated thatimmune ignorance can be overcome by delivering neoantigens to activateddendritic cells via antibody-tumor immune complexes. In these studies,simultaneous delivery of tumor binding antibodies and dendritic celladjuvants via intratumoral injections resulted in robust anti-tumorimmunity. New compositions and methods for the delivery of antibodiesand dendritic cell adjuvants are needed in order to reach inaccessibletumors and/or to expand treatment options for cancer patients and othersubjects. The invention provides such compositions and methods.

BRIEF SUMMARY OF THE INVENTION

The invention provides an immunoconjugate of formula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10, subscript n is an integer from about 2 to about25, and “Ab” is an antibody construct that has an antigen binding domainthat binds the protein human epidermal growth factor receptor 2(“HER2”).

The invention provides a composition comprising a plurality ofimmunoconjugates described herein.

The invention provides a method for treating cancer in a subjectcomprising administering a therapeutically effective amount of animmunoconjugate or a composition described herein to a subject in needthereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the HCC1954 human ductal carcinomatumor cell line. Median fluorescence intensity of co-stimulatorymolecule CD40 (cells gated on viable CD45+CD11c+HLA-DR+) was measured byflow cytometry and is shown for trastuzumab (dotted line, circle),trastuzumab+Compound 7 (dashed line, triangle) or Immunoconjugate A(solid line, square).

FIG. 1B shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the HCC1954 human ductal carcinomatumor cell line. Median fluorescence intensity of co-stimulatorymolecule CD86 (cells gated on viable CD45+CD11c+HLA-DR+) was measured byflow cytometry and is shown for trastuzumab (dotted line, circle),trastuzumab+Compound 7 (dashed line, triangle) or Immunoconjugate A(solid line, square).

FIG. 1C shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the HCC1954 human ductal carcinomatumor cell. TNFα secretion was measured by cytokine bead array (cellsgated on viable CD45+CD11c+HLA-DR+) for trastuzumab (dotted line,circle), trastuzumab+Compound 7 (dashed line, triangle) orImmunoconjugate A (solid line, square).

FIG. 1D shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the JIMT-1 human ductal carcinomatumor cell line. Median fluorescence intensity of co-stimulatorymolecule CD40 (cells gated on viable CD45+CD11c+HLA-DR+) was measured byflow cytometry and is shown for trastuzumab (dotted line, circle),trastuzumab+Compound 7 (dashed line, triangle) or Immunoconjugate A(solid line, square).

FIG. 1E shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the JIMT-1 human ductal carcinomatumor cell line. Median fluorescence intensity of co-stimulatorymolecule CD86 (cells gated on viable CD45+CD11c+HLA-DR+) was measured byflow cytometry and is shown for trastuzumab (dotted line, circle),trastuzumab+Compound 7 (dashed line, triangle) or Immunoconjugate A(solid line, square).

FIG. 1F shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the JIMT-1 human ductal carcinomatumor cell. TNFα secretion was measured by cytokine bead array (cellsgated on viable CD45+CD11c+HLA-DR+) for trastuzumab (dotted line,circle), trastuzumab+Compound 7 (dashed line, triangle) orImmunoconjugate A (solid line, square).

FIG. 1G shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the COLO 205 human colonadenocarcinoma cell line. Median fluorescence intensity ofco-stimulatory molecule CD40 (cells gated on viable CD45+CD11c+HLA-DR+)was measured by flow cytometry and is shown for trastuzumab (dottedline, circle), trastuzumab+Compound 7 (dashed line, triangle) orImmunoconjugate A (solid line, square).

FIG. 1H shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the COLO 205 human colonadenocarcinoma cell line. Median fluorescence intensity ofco-stimulatory molecule CD86 (cells gated on viable CD45+CD11c+HLA-DR+)was measured by flow cytometry and is shown for trastuzumab (dottedline, circle), trastuzumab+Compound 7 (dashed line, triangle) orImmunoconjugate A (solid line, square).

FIG. 1I shows the effect of Immunoconjugate A on myeloid activation inmyeloid APC-tumor co-cultures, using the COLO 205 human colonadenocarcinoma cell line. TNFα secretion was measured by cytokine beadarray (cells gated on viable CD45+CD11c+HLA-DR+) for trastuzumab (dottedline, circle), trastuzumab+Compound 7 (dashed line, triangle) orImmunoconjugate A (solid line, square).

FIG. 2A shows that Immunoconjugate B elicits myeloid differentiation asindicated by CD14 downregulation.

FIG. 2B shows that Immunoconjugate B elicits myeloid activation asindicated by CD40 upregulation.

FIG. 2C shows that Immunoconjugate B elicits myeloid activation asindicated by CD86 upregulation.

FIG. 2D shows TNFα secretion from myeloid cells following an 18 hourincubation with Immunoconjugate B.

FIG. 3A shows that Immunoconjugate C elicits myeloid differentiation asindicated by CD14 downregulation.

FIG. 3B shows that Immunoconjugate C elicits myeloid activation asindicated by CD40 upregulation.

FIG. 3C shows that Immunoconjugate C elicits myeloid activation asindicated by CD86 upregulation.

FIG. 3D shows TNFα secretion from myeloid cells following an 18 hourincubation with Immunoconjugate C.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an immunoconjugate of formula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10, subscript n is an integer from about 2 to about25, and “Ab” is an antibody construct that has an antigen binding domainthat binds human epidermal growth factor receptor 2 (“HER2”).

Antibody-adjuvant immunoconjugates of the invention, comprising anantibody construct that has an antigen binding domain that binds HER2linked to one or more adjuvant of formula:

demonstrate superior pharmacological properties over conventionalantibody conjugates. The polyethylene glycol-based linker (“PEG linker”)is the preferred linker to provide adequate purification and isolationof the immunoconjugate, maintain function of the one or more adjuvantmoieties and antibody construct, and produce ideal pharmacokinetic(“PK”) properties of the immunoconjugate. Additional embodiments andbenefits of the inventive antibody-adjuvant immunoconjugates will beapparent from description herein.

Definitions

As used herein, the term “immunoconjugate” refers to an antibodyconstruct that is covalently bonded to an adjuvant moiety via a linker.

As used herein, the phrase “antibody construct” refers to an antibody ora fusion protein comprising (i) an antigen binding domain and (ii) an Fcdomain.

As used herein, the term “antibody” refers to a polypeptide comprisingan antigen binding region (including the complementarity determiningregion (CDRs)) from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes HER2.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa) connected by disulfide bonds. Each chainis composed of structural domains, which are referred to asimmunoglobulin domains. These domains are classified into differentcategories by size and function, e.g., variable domains or regions onthe light and heavy chains (V_(L) and V_(H), respectively) and constantdomains or regions on the light and heavy chains (C_(L) and C_(H),respectively). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids, referred to as the paratope,primarily responsible for antigen recognition, i.e., the antigen bindingdomain. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. IgG antibodies are large molecules of about 150 kDacomposed of four peptide chains. IgG antibodies contain two identicalclass γ heavy chains of about 50 kDa and two identical light chains ofabout 25 kDa, thus a tetrameric quaternary structure. The two heavychains are linked to each other and to a light chain each by disulfidebonds. The resulting tetramer has two identical halves, which togetherform the Y-like shape. Each end of the fork contains an identicalantigen binding domain. There are four IgG subclasses (IgG1, IgG2, IgG3,and IgG4) in humans, named in order of their abundance in serum (i.e.,IgG1 is the most abundant). Typically, the antigen binding domain of anantibody will be most critical in specificity and affinity of binding tocancer cells.

Antibodies can exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into a Fab′ monomer. The Fab′ monomer is essentially Fab with partof the hinge region (see, e.g., Fundamental Immunology (Paul, editor,7th edition, 2012)). While various antibody fragments are defined interms of the digestion of an intact antibody, such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv), or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature, 348: 552-554(1990)).

The term “antibody” specifically encompasses monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments that exhibit the desired biological activity.

As used herein, the term “epitope” means any antigenic determinant orepitopic determinant of an antigen to which an antigen binding domainbinds (i.e., at the paratope of the antigen binding domain). Antigenicdeterminants usually consist of chemically active surface groupings ofmolecules, such as amino acids or sugar side chains, and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

As used herein, “HER2” refers to the protein human epidermal growthfactor receptor 2 (SEQ ID NO: 1), or an antigen with least about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more sequence identity to SEQ ID NO: 1.

Percent (%) identity of sequences can be calculated, for example, as100×[(identical positions)/min(TG_(A), TG_(B))], where TG_(A) and TG_(B)are the sum of the number of residues and internal gap positions inpeptide sequences A and B in the alignment that minimizes TG_(A) andTG_(B). See, e.g., Russell et al., J. Mol Biol., 244: 332-350 (1994).

As used herein, the term “adjuvant” refers to a substance capable ofeliciting an immune response in a subject exposed to the adjuvant. Thephrase “adjuvant moiety” refers to an adjuvant that is covalently bondedto an antibody construct, e.g., through a linker, as described herein.The adjuvant moiety can elicit the immune response while bonded to theantibody construct or after cleavage (e.g., enzymatic cleavage) from theantibody construct following administration of an immunoconjugate to thesubject.

As used herein, the terms “Toll-like receptor” and “TLR” refer to anymember of a family of highly-conserved mammalian proteins whichrecognizes pathogen-associated molecular patterns and acts as keysignaling elements in innate immunity. TLR polypeptides share acharacteristic structure that includes an extracellular domain that hasleucine-rich repeats, a transmembrane domain, and an intracellulardomain that is involved in TLR signaling.

The terms “Toll-like receptor 7” and “TLR7” refer to nucleic acids orpolypeptides sharing at least about 70%, about 80%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or more sequenceidentity to a publicly-available TLR7 sequence, e.g., GenBank accessionnumber AAZ99026 for human TLR7 polypeptide, or GenBank accession numberAAK62676 for murine TLR7 polypeptide.

The terms “Toll-like receptor 8” and “TLR8” refer to nucleic acids orpolypeptides sharing at least about 70%, about 80%, about 90%, about95%, about 96%, about 97%, about 98%, about 99%, or more sequenceidentity to a publicly-available TLR7 sequence, e.g., GenBank accessionnumber AAZ95441 for human TLR8 polypeptide, or GenBank accession numberAAK62677 for murine TLR8 polypeptide.

A “TLR agonist” is a substance that binds, directly or indirectly, to aTLR (e.g., TLR7 and/or TLR8) to induce TLR signaling. Any detectabledifference in TLR signaling can indicate that an agonist stimulates oractivates a TLR. Signaling differences can be manifested, for example,as changes in the expression of target genes, in the phosphorylation ofsignal transduction components, in the intracellular localization ofdownstream elements such as nuclear factor-κB (NF-κB), in theassociation of certain components (such as IL-1 receptor associatedkinase (IRAK)) with other proteins or intracellular structures, or inthe biochemical activity of components such as kinases (such asmitogen-activated protein kinase (MAPK)).

As used herein, “Ab” refers to an antibody construct that has anantigen-binding domain that binds HER2 (e.g., trastuzumab (also known asHERCEPTIN™), a biosimilar thereof, or a biobetter thereof.

As used herein, the term “biosimilar” refers to an approved antibodyconstruct that has active properties similar to the antibody constructpreviously approved (e.g., trastuzumab).

As used herein, the term “biobetter” refers to an approved antibodyconstruct that is an improvement of a previously approved antibodyconstruct (e.g., trastuzumab). The biobetter can have one or moremodifications (e.g., an altered glycan profile, or a unique epitope)over the previously approved antibody construct.

As used herein, the term “amino acid” refers to any monomeric unit thatcan be incorporated into a peptide, polypeptide, or protein. Amino acidsinclude naturally-occurring α-amino acids and their stereoisomers, aswell as unnatural (non-naturally occurring) amino acids and theirstereoisomers. “Stereoisomers” of a given amino acid refer to isomershaving the same molecular formula and intramolecular bonds but differentthree-dimensional arrangements of bonds and atoms (e.g., an L-amino acidand the corresponding D-amino acid). The amino acids can be glycosylated(e.g., N-linked glycans, O-linked glycans, phosphoglycans, C-linkedglycans, or glypiation) or deglycosylated.

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.Naturally-occurring α-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof. Stereoisomers of naturally-occurring α-amino acidsinclude, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys),D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine(D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg),D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine(D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser),D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine(D-Tyr), and combinations thereof.

Unnatural (non-naturally occurring) amino acids include, withoutlimitation, amino acid analogs, amino acid mimetics, synthetic aminoacids, N-substituted glycines, and N-methyl amino acids in either the L-or D-configuration that function in a manner similar to thenaturally-occurring amino acids. For example, “amino acid analogs” canbe unnatural amino acids that have the same basic chemical structure asnaturally-occurring amino acids (i.e., a carbon that is bonded to ahydrogen, a carboxyl group, an amino group) but have modified side-chaingroups or modified peptide backbones, e.g., homoserine, norleucine,methionine sulfoxide, and methionine methyl sulfonium. “Amino acidmimetics” refer to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “linker” refers to a functional group thatcovalently bonds two or more moieties in a compound or material. Forexample, the linking moiety can serve to covalently bond an adjuvantmoiety to an antibody construct in an immunoconjugate.

As used herein, the terms “treat,” “treatment,” and “treating” refer toany indicia of success in the treatment or amelioration of an injury,pathology, condition (e.g., cancer), or symptom (e.g., cognitiveimpairment), including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the symptom,injury, pathology, or condition more tolerable to the patient; reductionin the rate of symptom progression; decreasing the frequency or durationof the symptom or condition; or, in some situations, preventing theonset of the symptom. The treatment or amelioration of symptoms can bebased on any objective or subjective parameter, including, for example,the result of a physical examination.

The terms “cancer,” “neoplasm,” and “tumor” are used herein to refer tocells which exhibit autonomous, unregulated growth, such that the cellsexhibit an aberrant growth phenotype characterized by a significant lossof control over cell proliferation. Cells of interest for detection,analysis, and/or treatment in the context of the invention includecancer cells (e.g., cancer cells from an individual with cancer),malignant cancer cells, pre-metastatic cancer cells, metastatic cancercells, and non-metastatic cancer cells. Cancers of virtually everytissue are known. The phrase “cancer burden” refers to the quantum ofcancer cells or cancer volume in a subject. Reducing cancer burdenaccordingly refers to reducing the number of cancer cells or the cancercell volume in a subject. The term “cancer cell” as used herein refersto any cell that is a cancer cell (e.g., from any of the cancers forwhich an individual can be treated, e.g., isolated from an individualhaving cancer) or is derived from a cancer cell, e.g., clone of a cancercell. For example, a cancer cell can be from an established cancer cellline, can be a primary cell isolated from an individual with cancer, canbe a progeny cell from a primary cell isolated from an individual withcancer, and the like. In some embodiments, the term can also refer to aportion of a cancer cell, such as a sub-cellular portion, a cellmembrane portion, or a cell lysate of a cancer cell. Many types ofcancers are known to those of skill in the art, including solid tumorssuch as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, andmyelomas, and circulating cancers such as leukemias.

As used herein, the term “cancer” includes any form of cancer, includingbut not limited to, solid tumor cancers (e.g., lung, prostate, breast,gastric, bladder, colon, ovarian, pancreas, kidney, liver, glioblastoma,medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas,melanomas, and neuroendocrine) and liquid cancers (e.g., hematologicalcancers); carcinomas; soft tissue tumors; sarcomas; teratomas;melanomas; leukemias; lymphomas; and brain cancers, including minimalresidual disease, and including both primary and metastatic tumors. AnyHER2 expressing cancer is a suitable cancer to be treated by the subjectmethods and compositions. As used herein “HER2 expression” refers to acell that has a HER2 receptor on the cell's surface. For example, a cellmay have from about 20,000 to about 50,000 HER2 receptors on the cell'ssurface. As used herein “HER2 overexpression” refers to a cell that hasmore than about 50,000 HER2 receptors. For example, a cell 2, 5, 10,100, 1,000, 10,000, 100,000, or 1,000,000 times the number of HER2receptors as compared to corresponding non-cancer cell (e.g., about 1 or2 million HER2 receptors). It is estimated that HER2 is overexpressed inabout 25% to about 30% of breast cancers.

Carcinomas are malignancies that originate in the epithelial tissues.Epithelial cells cover the external surface of the body, line theinternal cavities, and form the lining of glandular tissues. Examples ofcarcinomas include, but are not limited to, adenocarcinoma (cancer thatbegins in glandular (secretory) cells such as cancers of the breast,pancreas, lung, prostate, stomach, gastroesophageal junction, and colon)adrenocortical carcinoma; hepatocellular carcinoma; renal cellcarcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma;carcinoma of the breast; basal cell carcinoma; squamous cell carcinoma;transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma;multilocular cystic renal cell carcinoma; oat cell carcinoma; large celllung carcinoma; small cell lung carcinoma; non-small cell lungcarcinoma; and the like. Carcinomas may be found in prostrate, pancreas,colon, brain (usually as secondary metastases), lung, breast, and skin.

Soft tissue tumors are a highly diverse group of rare tumors that arederived from connective tissue. Examples of soft tissue tumors include,but are not limited to, alveolar soft part sarcoma; angiomatoid fibroushistiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma;extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplasticsmall round-cell tumor; dermatofibrosarcoma protuberans; endometrialstromal tumor; Ewing's sarcoma; fibromatosis (Desmoid); fibrosarcoma,infantile; gastrointestinal stromal tumor; bone giant cell tumor;tenosynovial giant cell tumor; inflammatory myofibroblastic tumor;uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindlecell or pleomorphic lipoma; atypical lipoma; chondroid lipoma;well-differentiated liposarcoma; myxoid/round cell liposarcoma;pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma;high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignantperipheral nerve sheath tumor; mesothelioma; neuroblastoma;osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolarrhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignantschwannoma; synovial sarcoma; Evan's tumor; nodular fasciitis;desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcomaprotuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma;tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis(PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovialsarcoma; malignant peripheral nerve sheath tumor; neurofibroma;pleomorphic adenoma of soft tissue; and neoplasias derived fromfibroblasts, myofibroblasts, histiocytes, vascular cells/endothelialcells, and nerve sheath cells.

A sarcoma is a rare type of cancer that arises in cells of mesenchymalorigin, e.g., in bone or in the soft tissues of the body, includingcartilage, fat, muscle, blood vessels, fibrous tissue, or otherconnective or supportive tissue. Different types of sarcoma are based onwhere the cancer forms. For example, osteosarcoma forms in bone,liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examplesof sarcomas include, but are not limited to, askin's tumor; sarcomabotryoides; chondrosarcoma; ewing's sarcoma; malignanthemangioendothelioma; malignant schwannoma; osteosarcoma; and softtissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma;cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoidtumor; desmoplastic small round cell tumor; epithelioid sarcoma;extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma;gastrointestinal stromal tumor (GIST); hemangiopericytoma;hemangiosarcoma (more commonly referred to as “angiosarcoma”); kaposi'ssarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignantperipheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovialsarcoma; and undifferentiated pleomorphic sarcoma).

A teratoma is a type of germ cell tumor that may contain severaldifferent types of tissue (e.g., can include tissues derived from anyand/or all of the three germ layers: endoderm, mesoderm, and ectoderm),including, for example, hair, muscle, and bone. Teratomas occur mostoften in the ovaries in women, the testicles in men, and the tailbone inchildren.

Melanoma is a form of cancer that begins in melanocytes (cells that makethe pigment melanin). Melanoma may begin in a mole (skin melanoma), butcan also begin in other pigmented tissues, such as in the eye or in theintestines.

Leukemias are cancers that start in blood-forming tissue, such as thebone marrow, and cause large numbers of abnormal blood cells to beproduced and enter the bloodstream. For example, leukemias can originatein bone marrow-derived cells that normally mature in the bloodstream.Leukemias are named for how quickly the disease develops and progresses(e.g., acute versus chronic) and for the type of white blood cell thatis affected (e.g., myeloid versus lymphoid). Myeloid leukemias are alsocalled myelogenous or myeloblastic leukemias. Lymphoid leukemias arealso called lymphoblastic or lymphocytic leukemia. Lymphoid leukemiacells may collect in the lymph nodes, which can become swollen. Examplesof leukemias include, but are not limited to, Acute myeloid leukemia(AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia(CML), and Chronic lymphocytic leukemia (CLL).

Lymphomas are cancers that begin in cells of the immune system. Forexample, lymphomas can originate in bone marrow-derived cells thatnormally mature in the lymphatic system. There are two basic categoriesof lymphomas. One category of lymphoma is Hodgkin lymphoma (HL), whichis marked by the presence of a type of cell called the Reed-Sternbergcell. There are currently 6 recognized types of HL. Examples of Hodgkinlymphomas include nodular sclerosis classical Hodgkin lymphoma (CHL),mixed cellularity CHL, lymphocyte-depletion CHL, lymphocyte-rich CHL,and nodular lymphocyte predominant HL.

The other category of lymphoma is non-Hodgkin lymphomas (NHL), whichincludes a large, diverse group of cancers of immune system cells.Non-Hodgkin lymphomas can be further divided into cancers that have anindolent (slow-growing) course and those that have an aggressive(fast-growing) course. There are currently 61 recognized types of NHL.Examples of non-Hodgkin lymphomas include, but are not limited to,AIDS-related Lymphomas, anaplastic large-cell lymphoma,angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt'slymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma),chronic lymphocytic leukemia/small lymphocytic lymphoma, cutaneousT-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Celllymphoma, follicular lymphoma, hepatosplenic gamma-delta T-Celllymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle celllymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatriclymphoma, peripheral T-Cell lymphomas, primary central nervous systemlymphoma, transformed lymphomas, treatment-related T-Cell lymphomas, andWaldenstrom's macroglobulinemia.

Brain cancers include any cancer of the brain tissues. Examples of braincancers include, but are not limited to, gliomas (e.g., glioblastomas,astrocytomas, oligodendrogliomas, ependymomas, and the like),meningiomas, pituitary adenomas, and vestibular schwannomas, primitiveneuroectodermal tumors (medulloblastomas).

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, and invasion of surrounding or distant tissues or organs,such as lymph nodes.

As used herein, the phrases “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs, therefore, tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part that isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

As used herein the phrases “effective amount” and “therapeuticallyeffective amount” refer to a dose of a substance such as animmunoconjugate that produces therapeutic effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11^(th)Edition (McGraw-Hill, 2006); and Remington: The Science and Practice ofPharmacy, 22^(nd) Edition, (Pharmaceutical Press, London, 2012)).

As used herein, the terms “recipient,” “individual,” “subject,” “host,”and “patient” are used interchangeably and refer to any mammaliansubject for whom diagnosis, treatment, or therapy is desired (e.g.,humans). “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep,goats, pigs, camels, etc. In certain embodiments, the mammal is human.

The phrase “synergistic adjuvant” or “synergistic combination” in thecontext of this invention includes the combination of two immunemodulators such as a receptor agonist, cytokine, and adjuvantpolypeptide, that in combination elicit a synergistic effect on immunityrelative to either administered alone. Particularly, theimmunoconjugates disclosed herein comprise synergistic combinations ofthe claimed adjuvant and antibody construct. These synergisticcombinations upon administration elicit a greater effect on immunity,e.g., relative to when the antibody construct or adjuvant isadministered in the absence of the other moiety. Further, a decreasedamount of the immunoconjugate may be administered (as measured by thetotal number of antibody constructs or the total number of adjuvantsadministered as part of the immunoconjugate) compared to when either theantibody construct or adjuvant is administered alone.

As used herein, the term “administering” refers to parenteral,intravenous, intraperitoneal, intramuscular, intratumoral,intralesional, intranasal, or subcutaneous administration, oraladministration, administration as a suppository, topical contact,intrathecal administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to the subject.

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding the numerical value. Thus, if“X” is the value, “about X” or “around X” indicates a value of from 0.9Xto 1.1X, e.g., from 0.95X to 1.05X or from 0.99X to 1.01X. A referenceto “about X” or “around X” specifically indicates at least the values X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and1.05X. Accordingly, “about X” and “around X” are intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98×.”

Antibody Adjuvant Conjugates

The invention provides an immunoconjugate of formula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10, subscript n is an integer from about 2 to about 25(e.g., about 2 to about 16, about 6 to about 25, about 6 to about 16,about 8 to about 25, about 8 to about 16, about 6 to about 12, or about8 to about 12), and “Ab” is an antibody construct that has an antigenbinding domain that binds human epidermal growth factor receptor 2(“HER2”). “Ab” can be any suitable antibody construct that has anantigen binding domain that binds HER2, such as, for example,trastuzumab and pertuzumab. In certain embodiments, “Ab” is trastuzumab(also known as HERCEPTIN™), a biosimilar thereof, or a biobetterthereof. For example, “Ab” can be MYL-14010, ABP 980, BCD-022, CT-P6,EG12014, HD201, ONS-1050, PF-05280014, Ontruzant, Saiputing, Herzuma, orHLX02. In preferred embodiments, “Ab” is trastuzumab (also known asHERCEPTIN™).

Generally, the immunoconjugates of the invention comprise about 1 toabout 10 adjuvants, each adjuvant linked via a PEG linker to theantibody construct, as designated with subscript “r.” Each of theadjuvants linked via a PEG linker to the antibody construct isconjugated to the antibody construct at an amine of a lysine residue ofthe antibody construct. In an embodiment, r is 1, such that there is asingle adjuvant linked via a PEG linker to the antibody construct. Insome embodiments, r is an integer from about 2 to about 10 (e.g., about2 to about 9, about 3 to about 9, about 4 to about 9, about 5 to about9, about 6 to about 9, about 3 to about 8, about 3 to about 7, about 3to about 6, about 4 to about 8, about 4 to about 7, about 4 to about 6,about 5 to about 6, about 1 to about 6, about 1 to about 4, about 2 toabout 4, or about 1 to about 3). Accordingly, the immunoconjugates canhave (i.e., subscript “r” can be) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10adjuvants linked via a PEG linker. In preferred embodiments, theimmunoconjugates have (i.e., subscript “r” can be) 1, 2, 3, or 4adjuvants linked via a PEG linker. The desirable adjuvant to antibodyconstruct ratio (i.e., the value of the subscript “r”) can be determinedby a skilled artisan depending on the desired effect of the treatment.

Generally, the immunoconjugates of the invention comprise about 2 toabout 25 (e.g., about 2 to about 16, about 6 to about 25, about 6 toabout 16, about 8 to about 25, about 8 to about 16, about 6 to about 12,or about 8 to about 12) ethylene glycol units, as designated withsubscript “n.” Accordingly, the immunoconjugates of the invention cancomprise at least 2 ethylene glycol groups (e.g., at least 3 ethyleneglycol groups, at least 4 ethylene glycol groups, at least 5 ethyleneglycol groups, at least 6 ethylene glycol groups, at least 7 ethyleneglycol groups, at least 8 ethylene glycol groups, at least 9 ethyleneglycol groups, or at least 10 ethylene glycol groups). Accordingly, theimmunoconjugate can comprise from about 2 to about 25 ethylene glycolunits, for example, from about 6 to about 25 ethylene glycol units, fromabout 6 to about 16 ethylene glycol units, from about 8 to about 25ethylene glycol units, from about 8 to about 16 ethylene glycol units,from about 8 to about 12 ethylene glycol units, or from about 8 to about12 ethylene glycol units. In certain embodiments, the immunoconjugatecomprises a di(ethylene glycol) group, a tri(ethylene glycol) group, atetra(ethylene glycol) group, 5 ethylene glycol groups, 6 ethyleneglycol groups, 7 ethylene glycol groups, 8 ethylene glycol groups, 9ethylene glycol groups, 10 ethylene glycol groups, 11 ethylene glycolgroups, 12 ethylene glycol groups, 13 ethylene glycol groups, 14ethylene glycol groups, 15 ethylene glycol groups, 16 ethylene glycolgroups, 24 ethylene glycol groups, or 25 ethylene glycol groups. Inpreferred embodiments, the immunoconjugate comprises 6 ethylene glycolgroups, 8 ethylene glycol groups, 10 ethylene glycol groups, or 12ethylene glycol groups (i.e., about 6 ethylene glycol groups to about 12ethylene glycol groups).

The PEG linker can be linked to the antibody construct that has anantigen binding domain that binds HER2 (e.g., trastuzumab, pertuzumab,biosimilars thereof, and biobetters thereof) via an amine of a lysineresidue of the antibody construct. Accordingly, the immunoconjugates ofthe invention can be represented by the following formula:

wherein

is an antibody construct that has an antigen binding domain that bindsHER2 with residue

representing a lysine residue of the antibody construct, wherein “

” represents a point of attachment to the linker.

The adjuvant can be linked via the PEG linker to any suitable residue ofthe antibody construct, but desirably is linked to any lysine residue ofthe antibody construct. For example, the adjuvant can be linked via thePEG linker to one or more of K103, K107, K149, K169, K183, and/or K188of the light chain of the antibody construct, as numbered using theKabat numbering system. Alternatively, or additionally, the adjuvant canbe linked via the PEG linker to one or more of K30, K43, K65, K76, K136,K216, K217, K225, K293, K320, K323, K337, K395, and/or K417 of the heavychain of the antibody construct, as numbered using the Kabat numberingsystem. Generally, the adjuvant is predominantly linked via the PEGlinker at K107 or K188 of the light chain of the antibody construct, orK30, K43, K65, or K417 of the heavy chain of the antibody construct. Incertain embodiments, the adjuvant is linked via the PEG linker at K188of the light chain of the antibody construct, and optionally one or moreother lysine residues of the antibody construct.

Immunoconjugates as described herein can provide an unexpectedlyincreased activation response of an antigen presenting cell (“APC”).This increased activation can be detected in vitro or in vivo. In someembodiments, the increased APC activation can be detected in the form ofa reduced time to achieve a specified level of APC activation. Forexample, in an in vitro assay, % APC activation can be achieved at anequivalent dose with an immunoconjugate within about 1%, about 10%,about 20%, about 30%, about 40%, or about 50% of the time required toobtain the same or similar percentage of APC activation with a mixtureof unconjugated antibody construct and adjuvant, under otherwiseidentical concentrations and conditions. In some embodiments, animmunoconjugate can activate APCs (e.g., dendritic cells) and/or NKcells in a reduced amount of time. For example, in some embodiments, amixture of unconjugated antibody construct and adjuvant can activateAPCs (e.g., dendritic cells) and/or NK cells and/or induce dendriticcell differentiation after incubation with the mixture for 2, 3, 4, 5,1-5, 2-5, 3-5, or 4-7 days, while, in contrast, immunoconjugatesdescribed herein can activate and/or induce differentiation within 4hours, 8 hours, 12 hours, 16 hours, or 1 day, under otherwise identicalconcentrations and conditions. Alternatively, the increased APCactivation can be detected in the form of a reduced concentration ofimmunoconjugate required to achieve an amount (e.g., percent APCs),level (e.g., as measured by a level of upregulation of a suitablemarker) or rate (e.g., as detected by a time of incubation required toactivate) of APC activation.

In some embodiments, the immunoconjugates of the invention provide morethan an about 5% increase in activity compared to a mixture ofunconjugated antibody construct and adjuvant, under otherwise identicalconditions. In other embodiments, the immunoconjugates of the inventionprovide more than an about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, or about 70% increase in activity compared to a mixture ofunconjugated antibody construct and adjuvant, under otherwise identicalconditions. The increase in activity can be assessed by any suitablemeans, many of which are known to those ordinarily skilled in the artand can include myeloid activation, assessment by cytokine secretion, ora combination thereof.

In some embodiments, the invention provides an immunoconjugate offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is an antibody construct that has anantigen binding domain that binds human epidermal growth factor receptor2 (“HER2”).

In certain embodiments, the invention provides an immunoconjugate offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is trastuzumab (also known as HERCEPTIN™),pertuzumab, biosimilars thereof, and biobetters thereof. For example,“Ab” can be MYL-14010, ABP 980, BCD-022, CT-P6, EG12014, HD201,ONS-1050, PF-05280014, Ontruzant, Saiputing, Herzuma, or HLX02.

In preferred embodiments, the invention provides an immunoconjugate offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is trastuzumab (also known as HERCEPTIN™)

Adjuvants

The immunoconjugate of the invention comprises an adjuvant moiety offormula:

wherein the dashed line (“

”) represents a point of attachment of the adjuvant moiety to thelinker.

The adjuvant moiety described herein is a TLR agonist.

Antigen Binding Domain and Fc Domain

The immunoconjugates of the invention comprise an antibody constructthat comprises an antigen binding domain that binds HER2. In someembodiments, the antibody construct further comprises an Fc domain. Incertain embodiments, the antibody construct is an antibody. In certainembodiments, the antibody construct is a fusion protein.

The antigen binding domain can be a single-chain variable regionfragment (scFv). A single-chain variable region fragment (scFv), whichis a truncated Fab fragment including the variable (V) domain of anantibody heavy chain linked to a V domain of a light antibody chain viaa synthetic peptide, can be generated using routine recombinant DNAtechnology techniques. Similarly, disulfide-stabilized variable regionfragments (dsFv) can be prepared by recombinant DNA technology.

An embodiment of the invention provides antibody construct or antigenbinding domain which specifically recognizes and binds to HER2 (SEQ IDNO: 1). The antibody construct or antigen binding domain may compriseone or more variable regions (e.g., two variable regions) of an antigenbinding domain of an anti-HER2 antibody, each variable region comprisinga CDR1, a CDR2, and a CDR3.

An embodiment of the invention provides an antibody construct or antigenbinding domain comprising the CDR regions of trastuzumab. In thisregard, the antibody construct or antigen binding domain may comprise afirst variable region comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 2 (CDR1 of first variable region), a CDR2comprising the amino acid sequence of SEQ ID NO: 3 (CDR2 of firstvariable region), and a CDR3 comprising the amino acid sequence of SEQID NO: 4 (CDR3 of first variable region), and a second variable regioncomprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 5(CDR1 of second variable region), a CDR2 comprising the amino acidsequence of SEQ ID NO: 6 (CDR2 of second variable region), and a CDR3comprising the amino acid sequence of SEQ ID NO: 7 (CDR3 of secondvariable region). In this regard, the antibody construct can comprise(i) all of SEQ ID NOs: 2-4, (ii) all of SEQ ID NOs: 5-7, or (iii) all ofSEQ ID NOs: 2-7. Preferably, the antibody construct or antigen bindingdomain comprises all of SEQ ID NOs: 2-7.

In an embodiment of the invention, the antibody construct or antigenbinding domain comprising the CDR regions of trastuzumab furthercomprises the framework regions of the trastuzumab. In this regard, theantibody construct or antigen binding domain comprising the CDR regionsof the trastuzumab further comprises the amino acid sequence of SEQ IDNO: 8 (framework region (“FR”) 1 of first variable region), the aminoacid sequence of SEQ ID NO: 9 (FR2 of first variable region), the aminoacid sequence of SEQ ID NO: 10 (FR3 of first variable region), the aminoacid sequence of SEQ ID NO: 11 (FR4 of first variable region), the aminoacid sequence of SEQ ID NO: 12 (FR1 of second variable region), theamino acid sequence of SEQ ID NO: 13 (FR2 of second variable region),the amino acid sequence of SEQ ID NO: 14 (FR3 of second variableregion), and the amino acid sequence of SEQ ID NO: 15 (FR4 of secondvariable region). In this regard, the antibody construct or antigenbinding domain can comprise (i) all of SEQ ID NOs: 2-4 and 8-11, (ii)all of SEQ ID NOs: 5-7 and 12-15; or (iii) all of SEQ ID NOs: 2-7 and8-15.

An embodiment of the invention provides an antibody construct or antigenbinding domain comprising one or both variable regions of trastuzumab.In this regard, the first variable region may comprise SEQ ID NO: 16.The second variable region may comprise SEQ ID NO: 17. Accordingly, inan embodiment of the invention, the antibody construct or antigenbinding domain comprises SEQ ID NO: 16, SEQ ID NO: 17, or both SEQ IDNOs: 16 and 17. Preferably, the polypeptide comprises both of SEQ IDNOs: 16-17.

An embodiment of the invention provides an antibody construct or antigenbinding domain comprising the CDR regions of pertuzumab. In this regard,the antibody construct or antigen binding domain may comprise a firstvariable region comprising a CDR1 comprising the amino acid sequence ofSEQ ID NO: 18 (CDR1 of first variable region), a CDR2 comprising theamino acid sequence of SEQ ID NO: 19 (CDR2 of first variable region),and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20 (CDR3 offirst variable region), and a second variable region comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 21 (CDR1 of secondvariable region), a CDR2 comprising the amino acid sequence of SEQ IDNO: 22 (CDR2 of second variable region), and a CDR3 comprising the aminoacid sequence of SEQ ID NO: 23 (CDR3 of second variable region). In thisregard, the antibody construct can comprise (i) all of SEQ ID NOs:18-20, (ii) all of SEQ ID NOs: 21-23, or (iii) all of SEQ ID NOs: 18-23.Preferably, the antibody construct or antigen binding domain comprisesall of SEQ ID NOs: 18-23.

In an embodiment of the invention, the antibody construct or antigenbinding domain comprising the CDR regions of pertuzumab furthercomprises the framework regions of the pertuzumab. In this regard, theantibody construct or antigen binding domain comprising the CDR regionsof the pertuzumab further comprises the amino acid sequence of SEQ IDNO: 24 (framework region (“FR”) 1 of first variable region), the aminoacid sequence of SEQ ID NO: 25 (FR2 of first variable region), the aminoacid sequence of SEQ ID NO: 26 (FR3 of first variable region), the aminoacid sequence of SEQ ID NO: 27 (FR4 of first variable region), the aminoacid sequence of SEQ ID NO: 28 (FR1 of second variable region), theamino acid sequence of SEQ ID NO: 29 (FR2 of second variable region),the amino acid sequence of SEQ ID NO: 30 (FR3 of second variableregion), and the amino acid sequence of SEQ ID NO: 31 (FR4 of secondvariable region). In this regard, the antibody construct or antigenbinding domain can comprise (i) all of SEQ ID NOs: 18-20 and 24-26, (ii)all of SEQ ID NOs: 21-23 and 27-31; or (iii) all of SEQ ID NOs: 18-21and 24-31.

An embodiment of the invention provides an antibody construct or antigenbinding domain comprising one or both variable regions of pertuzumab. Inthis regard, the first variable region may comprise SEQ ID NO: 32. Thesecond variable region may comprise SEQ ID NO: 33. Accordingly, in anembodiment of the invention, the antibody construct or antigen bindingdomain comprises SEQ ID NO: 32, SEQ ID NO: 33, or both SEQ ID NOs: 32and 33. Preferably, the polypeptide comprises both of SEQ ID NOs: 32-33.

Included in the scope of the embodiments of the invention are functionalvariants of the antibody constructs or antigen binding domain describedherein. The term “functional variant” as used herein refers to anantibody construct having an antigen binding domain with substantial orsignificant sequence identity or similarity to a parent antibodyconstruct or antigen binding domain, which functional variant retainsthe biological activity of the antibody construct or antigen bindingdomain of which it is a variant. Functional variants encompass, forexample, those variants of the antibody constructs or antigen bindingdomain described herein (the parent antibody construct or antigenbinding domain) that retain the ability to recognize target cellsexpressing HER2 to a similar extent, the same extent, or to a higherextent, as the parent antibody construct or antigen binding domain.

In reference to the antibody construct or antigen binding domain, thefunctional variant can, for instance, be at least about 30%, about 50%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% ormore identical in amino acid sequence to the antibody construct orantigen binding domain.

A functional variant can, for example, comprise the amino acid sequenceof the parent antibody construct or antigen binding domain with at leastone conservative amino acid substitution. Alternatively, oradditionally, the functional variants can comprise the amino acidsequence of the parent antibody construct or antigen binding domain withat least one non-conservative amino acid substitution. In this case, itis preferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. The non-conservative amino acid substitution may enhance thebiological activity of the functional variant, such that the biologicalactivity of the functional variant is increased as compared to theparent antibody construct or antigen binding domain.

Amino acid substitutions of the inventive antibody constructs or antigenbinding domains are preferably conservative amino acid substitutions.Conservative amino acid substitutions are known in the art, and includeamino acid substitutions in which one amino acid having certain physicaland/or chemical properties is exchanged for another amino acid that hasthe same or similar chemical or physical properties. For instance, theconservative amino acid substitution can be an acidic/negatively chargedpolar amino acid substituted for another acidic/negatively charged polaramino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chainsubstituted for another amino acid with a nonpolar side chain (e.g.,Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), abasic/positively charged polar amino acid substituted for anotherbasic/positively charged polar amino acid (e.g., Lys, His, Arg, etc.),an uncharged amino acid with a polar side chain substituted for anotheruncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr,Tyr, etc.), an amino acid with a beta-branched side-chain substitutedfor another amino acid with a beta-branched side-chain (e.g., Ile, Thr,and Val), an amino acid with an aromatic side-chain substituted foranother amino acid with an aromatic side chain (e.g., His, Phe, Trp, andTyr), etc.

The antibody construct or antigen binding domain can consist essentiallyof the specified amino acid sequence or sequences described herein, suchthat other components, e.g., other amino acids, do not materially changethe biological activity of the antibody construct or antigen bindingdomain functional variant.

The antibody constructs and antigen binding domains of embodiments ofthe invention (including functional portions and functional variants)can be of any length, i.e., can comprise any number of amino acids,provided that the antibody constructs (or functional portions orfunctional variants thereof) retain their biological activity, e.g., theability to specifically bind to HER2, detect cancer cells in a mammal,or treat or prevent cancer in a mammal, etc. For example, the antibodyconstruct or antigen binding domain can be about 50 to about 5,000 aminoacids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500,600, 700, 800, 900, 1,000, or more amino acids in length.

The antibody constructs and antigen binding domains of embodiments ofthe invention (including functional portions and functional variants ofthe invention) can comprise synthetic amino acids in place of one ormore naturally-occurring amino acids. Such synthetic amino acids areknown in the art, and include, for example, aminocyclohexane carboxylicacid, norleucine, α-amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

The antibody constructs of embodiments of the invention (includingfunctional portions and functional variants) can be glycosylated,amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclizedvia, e.g., a disulfide bridge, or converted into an acid addition saltand/or optionally dimerized or polymerized.

In some embodiments, the antibody construct is a monoclonal antibody ofa defined sub-class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, or IgA₂). Ifcombinations of antibodies are used, the antibodies can be from the samesubclass or from different subclasses. Typically, the antibody constructis an IgG₁ antibody. Various combinations of different subclasses, indifferent relative proportions, can be obtained by those of skill in theart. In some embodiments, a specific subclass or a specific combinationof different subclasses can be particularly effective at cancertreatment or tumor size reduction. Accordingly, some embodiments of theinvention provide immunoconjugates wherein the antibody is a monoclonalantibody. In some embodiments, the monoclonal antibody is a humanizedmonoclonal antibody.

In some embodiments, the antibody construct or antigen binding domainbinds to HER2 on a cancer or immune cell at a higher affinity than acorresponding HER2 antigen on a non-cancer cell. For example, theantibody construct or antigen binding domain may preferentiallyrecognize HER2 containing a polymorphism that is found on a cancer orimmune cell as compared to recognition of a corresponding wild-type HER2antigen on the non-cancer. In some embodiments, the antibody constructor antigen binding domain binds a cancer cell with greater avidity thana non-cancer cell. For example, the cancer cell can express a higherdensity of HER2, thereby providing for a higher affinity binding of amultivalent antibody to the cancer cell.

In some embodiments, the antibody construct or antigen binding domaindoes not significantly bind non-cancer antigens (e.g., the antibodybinds one or more non-cancer antigens with at least 10, 100, 1,000,10,000, 100,000, or 1,000,000-fold lower affinity (higher Kd) thanHER2). In some embodiments, the corresponding non-cancer cell is a cellof the same tissue or origin that is not hyperproliferative or otherwisecancerous. HER2 need not be specific to the cancer cell or even enrichedin cancer cells relative to other cells (e.g., HER2 can be expressed byother cells). Thus, in the phrase “an antibody construct thatspecifically binds to an antigen of a cancer cell,” the term“specifically” refers to the specificity of the antibody construct andnot to the uniqueness of the presence of HER2 in that particular celltype.

Modified Fc Region

In some embodiments, the antibodies in the immunoconjugates contain amodified Fc region, wherein the modification modulates the binding ofthe Fc region to one or more Fc receptors.

The terms “Fc receptor” or “FcR” refer to a receptor that binds to theFc region of an antibody. There are three main classes of Fc receptors:(1) FcγR which bind to IgG, (2) FcαR which binds to IgA, and (3) FcRwhich binds to IgE. The FcγR family includes several members, such asFcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), andFcγRIIIB (CD16B). The Fcγ receptors differ in their affinity for IgG andalso have different affinities for the IgG subclasses (e.g., IgG1, IgG2,IgG3, and IgG4).

In some embodiments, the antibodies in the immunoconjugates (e.g.,antibodies conjugated to at least two adjuvant moieties) contain one ormore modifications (e.g., amino acid insertion, deletion, and/orsubstitution) in the Fc region that results in modulated binding (e.g.,increased binding or decreased binding) to one or more Fc receptors(e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a),and/or FcγRIIIB (CD16b)) as compared to the native antibody lacking themutation in the Fc region. In some embodiments, the antibodies in theimmunoconjugates contain one or more modifications (e.g., amino acidinsertion, deletion, and/or substitution) in the Fc region that reducethe binding of the Fc region of the antibody to FcγRIIB. In someembodiments, the antibodies in the immunoconjugates contain one or moremodifications (e.g., amino acid insertion, deletion, and/orsubstitution) in the Fc region of the antibody that reduce the bindingof the antibody to FcγRIIB while maintaining the same binding or havingincreased binding to FcγRI (CD64), FcγRIIA (CD32A), and/or FcRγIIIA(CD16a) as compared to the native antibody lacking the mutation in theFc region. In some embodiments, the antibodies in the immunoconjugatescontain one of more modifications in the Fc region that increase thebinding of the Fc region of the antibody to FcγRIIB.

In some embodiments, the modulated binding is provided by mutations inthe Fc region of the antibody relative to the native Fc region of theantibody. The mutations can be in a CH2 domain, a CH3 domain, or acombination thereof. A “native Fc region” is synonymous with a“wild-type Fc region” and comprises an amino acid sequence that isidentical to the amino acid sequence of an Fc region found in nature oridentical to the amino acid sequence of the Fc region found in thenative antibody (e.g., trastuzumab). Native sequence human Fc regionsinclude a native sequence human IgG1 Fc region, native sequence humanIgG2 Fc region, native sequence human IgG3 Fc region, and nativesequence human IgG4 Fc region, as well as naturally occurring variantsthereof. Native sequence Fc includes the various allotypes of Fcs (see,e.g., Jefferis et al., mAbs, 1(4): 332-338 (2009)).

In some embodiments, the mutations in the Fc region that result inmodulated binding to one or more Fc receptors can include one or more ofthe following mutations: SD (S239D), SDIE (S239D/I332E), SE (S267E),SELF (S267E/L328F), SDIE (S239D/I332E), SDIEAL (S239D/I332E/A330L), GA(G236A), ALIE (A330L/I332E), GASDALIE (G236A/S239D/A330L/I332E), V9(G237D/P238D/P271G/A330R), and V11 (G237D/P238D/H268D/P271G/A330R),and/or one or more mutations at the following amino acids: E233, G237,P238, H268, P271, L328 and A330. Additional Fc region modifications formodulating Fc receptor binding are described in, for example, U.S.Patent Application Publication 2016/0145350 and U.S. Pat. Nos. 7,416,726and 5,624,821, which are hereby incorporated by reference in theirentireties.

In some embodiments, the Fc region of the antibodies of theimmunoconjugates are modified to have an altered glycosylation patternof the Fc region compared to the native non-modified Fc region.

Human immunoglobulin is glycosylated at the Asn297 residue in the Cγ2domain of each heavy chain. This N-linked oligosaccharide is composed ofa core heptasaccharide, N-acetylglucosamine4Mannose3 (GlcNAc4Man3).Removal of the heptasaccharide with endoglycosidase or PNGase F is knownto lead to conformational changes in the antibody Fc region, which cansignificantly reduce antibody-binding affinity to activating FcγR andlead to decreased effector function. The core heptasaccharide is oftendecorated with galactose, bisecting GlcNAc, fucose, or sialic acid,which differentially impacts Fc binding to activating and inhibitoryFcγR. Additionally, it has been demonstrated that α2,6-sialyationenhances anti-inflammatory activity in vivo, while defucosylation leadsto improved FcγRIIIa binding and a 10-fold increase inantibody-dependent cellular cytotoxicity and antibody-dependentphagocytosis. Specific glycosylation patterns, therefore, can be used tocontrol inflammatory effector functions.

In some embodiments, the modification to alter the glycosylation patternis a mutation. For example, a substitution at Asn297. In someembodiments, Asn297 is mutated to glutamine (N297Q). Methods forcontrolling immune response with antibodies that modulate FcγR-regulatedsignaling are described, for example, in U.S. Pat. No. 7,416,726 andU.S. Patent Application Publications 2007/0014795 and 2008/0286819,which are hereby incorporated by reference in their entireties.

In some embodiments, the antibodies of the immunoconjugates are modifiedto contain an engineered Fab region with a non-naturally occurringglycosylation pattern. For example, hybridomas can be geneticallyengineered to secrete afucosylated mAb, desialylated mAb ordeglycosylated Fc with specific mutations that enable increased FcRγIIIabinding and effector function. In some embodiments, the antibodies ofthe immunoconjugates are engineered to be afucosylated.

In some embodiments, the entire Fc region of an antibody construct inthe immunoconjugates is exchanged with a different Fc region, so thatthe Fab region of the antibody is conjugated to a non-native Fc region.For example, the Fab region of trastuzumab, which normally comprises anIgG1 Fc region, can be conjugated to IgG2, IgG3, IgG4, or IgA, or theFab region of nivolumab, which normally comprises an IgG4 Fc region, canbe conjugated to IgG1, IgG2, IgG3, IgA1, or IgG2. In some embodiments,the Fc modified antibody with a non-native Fc domain also comprises oneor more amino acid modification, such as the S228P mutation within theIgG4 Fc, that modulate the stability of the Fc domain described. In someembodiments, the Fc modified antibody with a non-native Fc domain alsocomprises one or more amino acid modifications described herein thatmodulate Fc binding to FcR.

In some embodiments, the modifications that modulate the binding of theFc region to FcR do not alter the binding of the Fab region of theantibody to its antigen when compared to the native non-modifiedantibody. In other embodiments, the modifications that modulate thebinding of the Fc region to FcR also increase the binding of the Fabregion of the antibody to its antigen when compared to the nativenon-modified antibody.

Linker

Some of the immunoconjugates disclosed herein can be easier to purifythan an immunoconjugate comprising the same adjuvant, the same antibodyconstruct, and a different PEG linker length (e.g., PEG6 to PEG12 vs.PEG2 or PEG25). Without wishing to be bound by any particular theory, itis believed that the PEG6 to PEG12 immunoconjugates described hereinprovide a good balance of hydrophobicity and hydrophilicity tofacilitate the purification process. Some of the immunoconjugatesdisclosed herein can be easier to solubilize than an immunoconjugatecomprising the same adjuvant, the same antibody construct, and adifferent PEG linker length (e.g., PEG6 to PEG12 vs. PEG2 or PEG25).Without wishing to be bound by any particular theory, it is believedthat the PEG6 to PEG12 immunoconjugate described herein provide a goodbalance of hydrophobicity and hydrophilicity to maintain solubility andbe effective under biological conditions. It is also believed that thePEG6 to PEG12 immunoconjugate include a desirable number PEG units toprovide enough hydrophobicity to be readily purified and/or isolated,while maintaining enough hydrophilicity to be easily solubilized. Inpreferred embodiments, the immunoconjugate comprises a PEG10 linker.

Immunoconjugate Composition

The invention provides a composition, e.g., a pharmaceuticallyacceptable composition or formulation, comprising a plurality ofimmunoconjugates as described herein and optionally a carrier therefor,e.g., a pharmaceutically acceptable carrier. The immunoconjugates can bethe same or different in the composition, i.e., the composition cancomprise immunoconjugates that have the same number of adjuvants linkedto the same positions on the antibody construct and/or immunoconjugatesthat have the same number of adjuvants linked to different positions onthe antibody construct, that have different numbers of adjuvants linkedto the same positions on the antibody construct, or that have differentnumbers of adjuvants linked to different positions on the antibodyconstruct.

As described herein, the adjuvant can be linked via the PEG linker toany suitable residue of the antibody construct, desirably to a lysineresidue of the antibody construct. Thus, for example, the compositioncan comprise a plurality of immunoconjugates, wherein, for eachimmunoconjugate, one or more adjuvants are linked via PEG linkers to oneor more lysine residues selected from K103, K107, K149, K169, K183, andK188 of the light chain of the antibody construct, and K30, K43, K65,K76, K136, K216, K217, K225, K293, K320, K323, K337, K395, and K417 ofthe heavy chain of the antibody construct, as numbered using the Kabatnumbering system. Without wishing to be bound by any particular theory,the composition generally has a distribution of conjugation sites suchthat there is an average adjuvant to antibody construct ratio with agiven profile of preferred conjugation sites. In some embodiments, atleast about 40% (e.g., at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or at least about 90%) of the sum totalof lysine linkages occur at K188 of the light chain of the antibodyconstruct.

A composition of immunoconjugates of the invention can have an averageadjuvant to antibody construct ratio of about 0.4, 0.6, 0.8, 1, 1.2,1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2,4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7, 7.2,7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9, 9.2, 9.4, 9.6, 9.8, or 10, orwithin a range bounded by any two of the aforementioned values. Askilled artisan will recognize that the number of adjuvant conjugated tothe antibody construct may vary from immunoconjugate to immunoconjugatein a composition comprising multiple immunoconjugates of the invention,and, thus, the adjuvant to antibody construct (e.g., antibody) ratio canbe measured as an average. The adjuvant to antibody construct (e.g.,antibody) ratio can be assessed by any suitable means, many of which areknown in the art.

In some embodiments, the composition further comprises one or morepharmaceutically acceptable excipients. For example, theimmunoconjugates of the invention can be formulated for parenteraladministration, such as IV administration or administration into a bodycavity or lumen of an organ. Alternatively, the immunoconjugates can beinjected intra-tumorally. Compositions for injection will commonlycomprise a solution of the immunoconjugate dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and an isotonic solution of oneor more salts such as sodium chloride, e.g., Ringer's solution. Inaddition, sterile fixed oils can conventionally be employed as a solventor suspending medium. For this purpose, any bland fixed oil can beemployed, including synthetic monoglycerides or diglycerides. Inaddition, fatty acids such as oleic acid can likewise be used in thepreparation of injectables. These compositions desirably are sterile andgenerally free of undesirable matter. These compositions can besterilized by conventional, well known sterilization techniques. Thecompositions can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like.

The composition can contain any suitable concentration of theimmunoconjugate. The concentration of the immunoconjugate in thecomposition can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the patient'sneeds. In certain embodiments, the concentration of an immunoconjugatein a solution formulation for injection will range from about 0.1% (w/w)to about 10% (w/w).

Methods of Using the Immunoconjugate

The invention provides a method for treating cancer. The method includescomprising administering a therapeutically effective amount of animmunoconjugate as described herein (e.g., as a composition as describedherein) to a subject in need thereof, e.g., a subject that has cancerand is in need of treatment for the cancer.

Trastuzumab and pertuzumab, biosimilars thereof, and biobetters thereofare known to be useful in the treatment of cancer, particularly breastcancer, especially HER2-overexpressing breast cancer, gastric cancer,especially HER2-overexpressing gastric cancer, and gastroesophagealjunction adenocarcinoma. The immunoconjugate described herein can beused to treat the same types of cancers as trastuzumab, pertuzumab,biosimilars thereof, and biobetters thereof particularly breast cancer,especially HER2-overexpressing breast cancer, gastric cancer, especiallyHER2-overexpressing gastric cancer, and gastroesophageal junctionadenocarcinoma.

The immunoconjugate is administered to a subject in need thereof in anytherapeutically effective amount using any suitable dosing regimen, suchas the dosing regimens utilized for trastuzumab, pertuzumab, biosimilarsthereof, and biobetters thereof. For example, the methods can includeadministering the immunoconjugate to provide a dose of from about 100ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose canrange from about 5 mg/kg to about 50 mg/kg, from about 10 μg/kg to about5 mg/kg, or from about 100 μg/kg to about 1 mg/kg. The immunoconjugatedose can be about 100, 200, 300, 400, or 500 μg/kg. The immunoconjugatedose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. Theimmunoconjugate dose can also be outside of these ranges, depending onthe particular conjugate as well as the type and severity of the cancerbeing treated. Frequency of administration can range from a single doseto multiple doses per week, or more frequently. In some embodiments, theimmunoconjugate is administered from about once per month to about fivetimes per week. In some embodiments, the immunoconjugate is administeredonce per week.

In another aspect, the invention provides a method for preventingcancer. The method comprises administering a therapeutically effectiveamount of an immunoconjugate (e.g., as a composition as described above)to a subject. In certain embodiments, the subject is susceptible to acertain cancer to be prevented. For example, the methods can includeadministering the immunoconjugate to provide a dose of from about 100ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose canrange from about 5 mg/kg to about 50 mg/kg, from about 10 μg/kg to about5 mg/kg, or from about 100 μg/kg to about 1 mg/kg. The immunoconjugatedose can be about 100, 200, 300, 400, or 500 μg/kg. The immunoconjugatedose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. Theimmunoconjugate dose can also be outside of these ranges, depending onthe particular conjugate as well as the type and severity of the cancerbeing treated. Frequency of administration can range from a single doseto multiple doses per week, or more frequently. In some embodiments, theimmunoconjugate is administered from about once per month to about fivetimes per week. In some embodiments, the immunoconjugate is administeredonce per week.

Some embodiments of the invention provide methods for treating cancer asdescribed above, wherein the cancer is breast cancer. Breast cancer canoriginate from different areas in the breast, and a number of differenttypes of breast cancer have been characterized. For example, theimmunoconjugates of the invention can be used for treating ductalcarcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma;medullary carcinoma; mucinous carcinoma; papillary carcinoma; orcribriform carcinoma of the breast); lobular carcinoma in situ; invasivelobular carcinoma; inflammatory breast cancer; and other forms of breastcancer. In some embodiments, methods for treating breast cancer includeadministering an immunoconjugate containing an antibody construct thatis capable of binding HER2 (e.g., trastuzumab, pertuzumab, biosimilarsthereof, and biobetters thereof).

Some embodiments of the invention provide methods for treating cancer asdescribed above, wherein the cancer is gastric cancer. Gastric (stomach)cancer can originate from different cells in the stomach and severaltypes of gastric cancer have been characterized includingadenocarcinoma, carcinoid tumors, squamous cell carcinoma, small cellcarcinoma, leiomyosarcoma, and gastrointestinal stromal tumors. In someembodiments, methods for treating gastric cancer include administeringan immunoconjugate containing an antibody construct that is capable ofbinding HER2 (e.g., trastuzumab).

Some embodiments of the invention provide methods for treating cancer asdescribed above, wherein the cancer is gastroesophageal junctioncarcinoma. This carcinoma occurs in the area where the esophagus meatsthe stomach. There are three types of gastroesophageal junctioncarcinoma. In Type 1, the cancer the cancer grows down from above andinto the gastroesophageal junction. The normal lining of the lower endof the esophagus is replaced by mutations (also called Barrett'sesophagus). In Type 2, the cancer grows at the gastroesophageal junctionby itself. In Type 3, the cancer grows up into the gastroesophagealjunction from the stomach upwards. In some embodiments, methods fortreating gastroesophageal junction carcinoma include administering animmunoconjugate containing an antibody construct that is capable ofbinding HER2 (e.g., trastuzumab).

In some embodiments, the cancer is susceptible to a pro-inflammatoryresponse induced by TLR7 and/or TLR8.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the invention described herein may bebeneficial alone or in combination, with one or more other aspects orembodiments. Without limiting the foregoing description, certainnon-limiting aspects of the disclosure numbered 1-33 are provided below.As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individually numbered aspects may be used orcombined with any of the preceding or following individually numberedaspects. This is intended to provide support for all such combinationsof aspects and is not limited to combinations of aspects explicitlyprovided below:

1. An immunoconjugate of formula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10, subscript n is an integer from about 2 to about25, and “Ab” is an antibody construct that has an antigen binding domainthat binds HER2.

2. The immunoconjugate of aspect 1, wherein subscript r is an integerfrom 1 to 6.

3. The immunoconjugate of aspect 2, wherein subscript r is an integerfrom 1 to 4.

4. The immunoconjugate of aspect 3, wherein subscript r is 1.

5. The immunoconjugate of aspect 3, wherein subscript r is 2.

6. The immunoconjugate of aspect 3, wherein subscript r is 3.

7. The immunoconjugate of aspect 3, wherein subscript r is 4.

8. The immunoconjugate of any one of aspects 1-7, wherein subscript n isan integer from 6 to 12.

9. The immunoconjugate of aspect 8, wherein subscript n is an integerfrom 8 to 12.

10. The immunoconjugate of aspect 1, wherein the immunoconjugate is offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is an antibody construct that has anantigen binding domain that binds HER2 (e.g., trastuzumab (also known asHERCEPTIN™), a biosimilar thereof, or a biobetter thereof).

11. The immunoconjugate of aspect 1, wherein the immunoconjugate is offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is an antibody construct that has anantigen binding domain that binds HER2 (e.g., trastuzumab (also known asHERCEPTIN™), a biosimilar thereof, or a biobetter thereof).

12. The immunoconjugate of aspect 1, wherein the immunoconjugate is offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is an antibody construct that has anantigen binding domain that binds HER2 (e.g., trastuzumab (also known asHERCEPTIN™), a biosimilar thereof, or a biobetter thereof).

13. The immunoconjugate of aspect 1, wherein the immunoconjugate is offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is an antibody construct that has anantigen binding domain that binds HER2 (e.g., trastuzumab (also known asHERCEPTIN™), a biosimilar thereof, or a biobetter thereof).

14. The immunoconjugate of aspect 1, wherein the immunoconjugate is offormula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10 and “Ab” is an antibody construct that has anantigen binding domain that binds HER2 (e.g., trastuzumab (also known asHERCEPTIN™), a biosimilar thereof, or a biobetter thereof).

15. The immunoconjugate of any one of aspects 1-14, wherein “Ab” istrastuzumab, a biosimilar thereof, or a biobetter thereof.

16. The immunoconjugate of any one of aspects 1-14, wherein “Ab” ispertuzumab, a biosimilar thereof, or a biobetter thereof.

17. The immunoconjugate of aspect 15, wherein “Ab” is trastuzumab.

18. The immunoconjugate of aspect 15, wherein “Ab” is a biosimilar oftrastuzumab.

19. A composition comprising a plurality of immunoconjugates accordingto any one of aspects 1-18.

20. The composition of aspect 19, wherein the average adjuvant toantibody construct ratio is from about 0.01 to about 10.

21. The composition of aspect 20, wherein the average adjuvant toantibody construct ratio is from about 1 to about 10.

22. The composition of aspect 21, wherein the average adjuvant toantibody construct ratio is from about 1 to about 6.

23. The composition of aspect 22, wherein the average adjuvant toantibody construct ratio is from about 1 to about 4.

24. The composition of aspect 23, wherein the average adjuvant toantibody construct ratio is from about 1 to about 3.

25. A method for treating cancer comprising administering atherapeutically effective amount of an immunoconjugate according to anyone of aspects 1-18 or a composition according to any one of aspects19-24 to a subject in need thereof.

26. The method of aspect 25, wherein the cancer is susceptible to apro-inflammatory response induced by TLR7 and/or TLR8 agonism.

27. The method of aspect 25 or 26, wherein the cancer is aHER2-expressing cancer.

28. The method of any one of aspects 25-27, wherein the cancer is breastcancer.

29. The method of aspect 28, wherein the breast cancer is HER2overexpressing breast cancer.

30. The method of any one of aspect 25-27, wherein the cancer is gastriccancer.

31. The method of aspect 30, wherein the gastric cancer is HER2overexpressing gastric cancer.

32. The method of any one of aspect 25-27, 30, or 31, wherein the canceris gastroesophageal junction adenocarcinoma.

33. Use of an immunoconjugate according to any one of aspect 1-18 or acomposition according to any one of aspects 19-24 for treating cancer.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1: Synthesis of Compound 2

To a solution of 6-bromo-2,4-dichloro-3-nitroquinoline (5.6 g, 17.4mmol, 1 eq.) and solid K₂CO₃ (3.6 g, 26 mmol, 1.5 eq.) indimethylformamide (DMF, 100 mL) at room temperature was added neat2,4-dimethoxybenzylamine (3.5 g, 20.1 mmol, 1.2 eq.). The mixture wasstirred for 15 minutes, water (300 mL) was added, and then the mixturewas stirred for 5 minutes. The resultant solid was filtered and thendissolved in ethyl acetate (100 mL). The solution was washed with water(100 mL), brine (100 mL), separated, dried (Na₂SO₄), then filtered andconcentrated in vacuo. The brown solid was triturated with 1:1hexanes/diethyl ether (150 mL) and filtered to obtain6-bromo-2-chloro-4-(2,4-dimethoxybenzyl)amino-3-nitroquinoline (6.9 g,15.3 mmol, 88%) as a yellow solid. The compound was used without furtherpurification.

Example 2: Synthesis of Compound 3

NiCl₂.6H₂O (0.36 g, 1.5 mmol, 0.1 eq.) was added to6-bromo-2-chloro-4-(2,4-dimethoxybenzyl)amino-3-nitroquinoline (6.9 g,15.3 mmol, 88%) in methanol (200 mL) at 0° C. Sodium borohydride(pellets, 1.42 g, 38 mmol, 2.5 eq.) was added and the reaction wasstirred for 1 h at 0° C. then warmed to room temperature and stirred foranother 15 minutes. Glacial acetic acid (5 mL) was added until a pH of˜5 was obtained. The solvent was evaporated in vacuo and the crude solidwas re-dissolved in ethyl acetate (150 mL) then filtered through a bedof diatomaceous earth to remove a black insoluble material. The ethylacetate was removed in vacuo. The dark brown solid was triturated withether (75 mL) then filtered to obtain3-amino-6-bromo-2-chloro-4-(2,4-dimethoxybenzyl)aminoquinoline (5.81 g,13.7 mmol, 90%) as a tan solid. The compound was used without furtherpurification.

Example 3: Synthesis of Compound 4

Neat valeroyl chloride (2.0 mL, 2.0 g, 16 mmol, 1.2 eq) was added to asolution of3-amino-6-bromo-2-chloro-4-(2,4-dimethoxybenzyl)aminoquinoline (5.75 g,13.6 mmol, 1 eq.) in dichloromethane (100 mL) containing triethylamine(2.1 g, 2.8 mL, 20 mmol, 1.5 eq.) while stirred at room temperature. Themixture was washed with water (150 mL), brine (150 mL), separated, thendried (Na₂SO₄), filtered, and concentrated. The solid was trituratedwith ether, filtered, and then dried under vacuum.N-(6-bromo-2-chloro-4-((2,4-dimethoxybenzyl)amino)quinolin-3-yl)pentanamidewas obtained as a brown solid (5.8 g, 11.4 mmol, 84%). The compound wasused without further purification.

Example 4: Synthesis of Compound

In a 100 mL beaker a mixture ofN-(6-bromo-2-chloro-4-((2,4-dimethoxybenzyl)amino)quinolin-3-yl)pentanamide(5.8 g, 11.4 mmol, 1 eq.) and 2-chlorobenzoic (0.90 g, 5.7 mmol. 0.5eq.) was boiled in 50 mL toluene for 2 hours. Toluene was added to 50 mLeach time the volume reached 25 mL. 2,4-dimethoxybenzylamine (9.5 g, 57mmol, 5 eq.) was added and the reaction was maintained at 120° C. for 2hours. The reaction was cooled to room temperature and water (80 mL)then acetic acid (3.5 mL) was added. The supernatant was decanted andthe crude product was washed with water (80 mL). The wet solid wastriturated with methanol (100 mL) to provide8-bromo-2-butyl-N,1-bis(2,4-dimethoxybenzyl)-1H-imidazo[4,5-c]quinolin-4-amine(4.80 g, 7.7 mmol, 68%) as an off-white solid. The compound was usedwithout further purification.

Example 5: Synthesis of Compound 6

A mixture of8-bromo-2-butyl-N,1-bis(2,4-dimethoxybenzyl)-1H-imidazo[4,5-c]quinolin-4-amine(0.31 g, 0.5 mmol, 1 eq.) and tert-butyl piperazine-1-carboxylate (0.19g, 1 mmol, 2 eq.) were combined in toluene (2 mL) then degassed withargon. Pd₂dba₃ (45 mg, 0.05 mmol, 0.1 eq.), tri-tert-butylphosphinetetrafluoroborate (29 mg, 0.10 mmol, 0.2 eq) and sodium tert-butoxide(144 mg, 1.5 mmol, 3 eq) were added. The mixture was heated in a cappedvial at 110° C. for 30 minutes. The mixture was cooled then partitionedbetween ethyl acetate (50 mL) and water (50 mL). The organic layer waswashed with brine (50 mL), dried with sodium sulfate, filtered andconcentrated in vacuo. The crude product was purified on silica gel (20g) and then eluted with 50% ethyl acetate/hexanes to yield tert-butyl4-(2-butyl-1-(2,4-dimethoxybenzyl)-4-((2,4-dimethoxybenzyl)amino)-1H-imidazo[4,5-c]quinolin-8-yl)piperazine-1-carboxylate(0.28 g, 0.39 mmol, 78%) as an off-white solid. LC/MS [M+H]725.40(calculated); LC/MS [M+H] 725.67 (observed).

Example 6: Synthesis of Compound 7

Tert-butyl4-(2-butyl-1-(2,4-dimethoxybenzyl)-4-((2,4-dimethoxybenzyl)amino)-1H-imidazo[4,5-c]quinolin-8-yl)piperazine-1-carboxylate(0.28 g, 0.39 mmol, 1 eq.) was dissolved in TFA (3 mL) and heated toreflux for 5 min. The TFA was removed in vacuo and the crude product wasdissolved in acetonitrile, filtered then concentrated to obtain the TFAsalt of 2-butyl-8-(piperazin-1-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.16 g, 0.37 mmol, 95%) as an off-white solid. LC/MS [M+H] 325.21(calculated); LC/MS [M+H] 325.51 (observed).

Example 7: Synthesis of Compound 8

In a 40 mL vial flushed with nitrogen, oxalyl chloride (1.84 g, 1.24 mL,14.5 mmol, 2.5 eq) was added then dichloromethane (10 mL). The solutionwas cooled to −78° C. A solution of DMSO (2.26 g, 2.05 mL, 29 mmol, 5eq) in dichloromethane (9 mL) was added dropwise and the mixture wasstirred for 15 minutes. A solution of tert-butyl1-hydroxy-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oate (3.4g, 5.8 mmol, 1 eq) in dichloromethane (9 mL) was added dropwise and themixture was stirred for 30 minutes at −78° C. Triethylamine (4.4 g, 6.0mL, 43.5 mmol, 7.5 eq) was added dropwise. This mixture was stirred for30 min at −78° C. then warmed to room temperature over 30 minutes. To a100 mL round bottom flask containing2-butyl-8-(piperazin-1-yl)-1H-imidazo[4,5-c]quinolin-4-aminehydrochloride (2.1 g, 5.8 mmol, 1 eq) and sodium triacetoxyborohydride(5.5 g, 26 mmol, 4.5 eq) in DMF (30 mL) was slowly added tert-butyl1-oxo-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oate(theoretical amount 5.8 mmol, 1 eq) and the reaction was stirred at roomtemperature for 1 hour. The dichloromethane was removed under reducedpressure, and then 20% Na₂CO₃ (20 mL) was added and the mixture wasstirred vigorously for 15 minutes. All of the solvent was removed andthe solid material was suspended and sonicated in 10%methanol/dichloromethane, then filtered through diatomaceous earth. Thefilter cake was washed with 10% methanol/dichloromethane and thecombined filtrates were concentrated. Purification by flashchromatography (80 g REDISEP™ gold silica column) was performed using a2-20% MeOH/dichloromethane+0.1% triethylamine (55 mL/min) gradient over28 min. The pure fractions were combined and concentrated to obtaintert-butyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oate(3.9 g, 4.4 mmol, 75%) as a slightly golden syrup. The impure fractionscontaining were re-purified then combined to give a final mass (4.26 g,4.8 mmol, 83%). LC/MS [M+H] 893.55 (calculated); LC/MS [M+H] 893.98(observed).

Example 8: Synthesis of Compound 9

Tert-butyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oate(4.26 g, 4.8 mmol) was dissolved in a 1:1 mixture of 3 M aq. HCl anddioxane (100 mL) and heated at 60° C. for 60 min. After hydrolysis wascomplete the solvent was removed under reduced pressure. The1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oicacid hydrochloride obtained was azeotroped 4 times with acetonitrile (75mL) then suspended in acetonitrile (75 mL) and centrifuged at 4000 rpmfor 4 minutes. This process was repeated. The solid was transferred to a100 mL round bottom flask with acetonitrile and concentrated by underreduced pressure to obtain a yellow, hygroscopic solid (4.0 g, 4.6 mmol,95%) that was used as is in the next reaction. LC/MS [M+H] 837.49(calculated); LC/MS [M+H] 837.84 (observed).

Example 9: Synthesis of Compound 10

To a 250 mL round bottom flask containing the1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oicacid hydrochloride (4.0 g, 4.6 mmol, 1 eq) was added a suspension of2,3,5,6-tetrafluorophenol (1.64 g, 10 mmol, 2.4 eq) and EDC (2.0 g, 11mmol, 2.3 eq.) in anhydrous DMF (50 mL) and the mixture was allowed tostir at room temperature for 30 minutes. The mixture was then heated at50° C. for 30 minutes. Most of the DMF (˜90%) was removed by azeotropingwith toluene (80 mL) under reduced pressure with the bath temperatureset to 50° C. To this crude material was added diethyl ether (100 mL)and the pasty solid was stirred vigorously. The supernatant wasdiscarded. This process was repeated. The crude material was dissolvedin 40 mL ethyl acetate/acetone/acetic acid/water (6:2:1:1). The crudesolution was divided into two equal portions and each was purified on a40 g REDISEP™ gold silica column (Teledyne Isco, Lincoln, Nebr.) usingisocratic eluent ethyl acetate/acetone/acetic acid/water (6:2:1:1) toobtain 2,3,5,6-tetrafluorophenyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oate(3.34 g, 3.4 mmol, 74%) as an orange paste. LC/MS [M+H]985.49(calculated); LC/MS [M+H] 985.71 (observed).

Example 10: Synthesis of Compound 11

2-butyl-8-(piperazin-1-yl)-1H-imidazo[4,5-c]quinolin-4-amine wasconverted into tert-butyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oateaccording to the procedure described in Example 7. LC/MS [M+H] 717.45(calculated); LC/MS [M+H] 717.75 (observed).

Example 11: Synthesis of Compound 12

Tert-butyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oatewas converted into1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oicacid according to the procedure described in Example 8. LC/MS [M+H]661.39 (calculated); LC/MS [M+H] 661.60 (observed).

Example 12: Synthesis of Compound 13

1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oicacid was converted into 2,3,5,6-tetrafluorophenyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oateaccording to the procedure described in Example 9. LC/MS [M+H] 809.39(calculated); LC/MS [M+H] 809.62 (observed).

Example 13: Synthesis of Compound 14

2-butyl-8-(piperazin-1-yl)-1H-imidazo[4,5-c]quinolin-4-amine wasconverted into tert-butyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oateaccording to the procedure described in Example 7. LC/MS [M+H] 981.61(calculated); LC/MS [M+H]981.86 (observed).

Example 14: Synthesis of Compound 15

Tert-butyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oatewas converted into1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid according to the procedure described in Example 8. Compound wasused without further purification.

Example 15: Synthesis of Compound 16

1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid was converted into 2,3,5,6-tetrafluorophenyl1-(4-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-8-yl)piperazin-1-yl)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oateaccording to the procedure described in Example 9. LC/MS [M+H] 1073.54(calculated); LC/MS [M+H]1073.81 (observed).

Example 16: Synthesis of Immunoconjugate A

This example demonstrates the synthesis of Immunoconjugate A withtrastuzumab as the antibody construct (Tras).

Trastuzumab was buffer exchanged into the conjugation buffer containing100 mM boric acid, 50 mM sodium chloride, 1 mMethylenediaminetetraacetic acid at pH 83, using G-25 SEPHADEX™ desaltingcolumns (Sigma-Aldrich, St. Louis, Mo.). The eluates were then eachadjusted to 6 mg/ml using the buffer and sterile filtered. Trastuzumabat 6 mg/ml was pre-warmed to 30° C. and rapidly mixed with 7 molarequivalents of Compound 10. The reaction was allowed to proceed for 16hours at 30° C. and Immunoconjugate A was separated from reactants byrunning over two successive G-25 desalting columns equilibrated inphosphate buffered saline at pH 7.2. Adjuvant-antibody ratios (DAR) wasdetermined by liquid chromatography mass spectrometry analysis using aC4 reverse phase column on an ACQUITY™ UPLC H-class (Waters Corporation,Milford, Mass.) connected to a XEVO™ G2-XS TOF mass spectrometer (WatersCorporation). Immunoconjugate A had a DAR of 2.5.

Example 17: Synthesis of Immunoconjugate B

This example demonstrates the synthesis of Immunoconjugate B withtrastuzumab as the antibody construct (Tras).

Trastuzumab was buffer exchanged into the conjugation buffer containing100 mM boric acid, 50 mM sodium chloride, 1 mMethylenediaminetetraacetic acid at pH 8.3, using C-25 SEPHADEX™desalting columns (Sigma-Aldrich). The eluates were then each adjustedto 6 mg/ml using the buffer and sterile filtered. Trastuzumab at 6 mg/mlwas pre-warmed to 30° C. and rapidly mixed with 8.5 molar equivalents ofCompound 13. The reaction was allowed to proceed for 16 hours at 30° C.and Immunoconjugate B was separated from reactants by running over twosuccessive G-25 SEPHADEX™ desalting columns (Sigma-Aldrich) equilibratedin phosphate buffered saline at pH 7.2. Adjuvant-antibody ratios (DAR)was determined by liquid chromatography mass spectrometry analysis usinga C4 reverse phase column on an ACQUITY™ UPLC H-class (WatersCorporation, Milford, Mass.) connected to a XEVO™ G2-XS TOF massspectrometer (Waters Corporation). Immunoconjugate B had a DAR of 2.37.

Example 18: Synthesis of Immunoconjugate C

This example demonstrates the synthesis of Immunoconjugate C withtrastuzumab as the antibody construct (Tras).

Trastuzumab was buffer exchanged into the conjugation buffer containing100 mM boric acid, 50 mM sodium chloride, 1 mMethylenediaminetetraacetic acid at pH 8.3, using G-25 SEPHADEX™desalting columns (Sigma-Aldrich). The eluates were then each adjustedto 6 mg/ml using the buffer and sterile filtered, Trastuzunab at 6mg/mil was pre-warmed to 30° C. and rapidly mixed with 6 molarequivalents of Compound 16. The reaction was allowed to proceed for 16hours at 30° C. and Immunoconjugate C was separated from reactants byrunning over two successive G-25 desalting columns equilibrated inphosphate buffered saline at pH 7.2. Adjuvant-antibody ratios (DAR) wasdetermined by liquid chromatography mass spectrometry analysis using aC4 reverse phase column on an ACQUITY™ UPLC H-class (Waters Corporation,Milford, Mass.) connected to a XEVO™ G2-XS TOF mass spectrometer (WatersCorporation). Immunoconjugate C had a DAR of 2.15.

Example 19. Assessment of Immunoconjugate Activity In Vitro

This example shows that Immunoconjugate A, Immunoconjugate B, andImmunoconjugate C are effective at eliciting myeloid activation, andtherefore are useful for the treatment of cancer.

Isolation of Human Antigen Presenting Cells. Human myeloid antigenpresenting cells (APCs) were negatively selected from human peripheralblood obtained from healthy blood donors (Stanford Blood Center, PaloAlto, Calif.) by density gradient centrifugation using a ROSETTESEP™Human Monocyte Enrichment Cocktail (Stem Cell Technologies, Vancouver,Canada) containing monoclonal antibodies against CD14, CD16, CD40, CD86,CD123, and HLA-DR. Immature APCs were subsequently purified to >97%purity via negative selection using an EASYSEP™ Human MonocyteEnrichment Kit (Stem Cell Technologies) without CD16 depletioncontaining monoclonal antibodies against CD14, CD16, CD40, CD86, CD123,and HLA-DR.

Preparation of Tumor Cells. Three tumor cell lines were used: HCC1954,JIMT-1, and COLO 205. HCC1954 (American Type Culture Collection (ATCC),Manassas, Va.) was derived from a primary stage IIA, grade 3 invasiveductal carcinoma with no lymph node metastases. HCC1954 is positive forthe epithelial cell specific marker Epithelial Glycoprotein 2 and forcytokeratin 19, and is negative for expression of estrogen receptor (ER)and progesterone receptor (PR). HCC1954 overexpresses HER2 (asdetermined by enzyme-linked immunosorbent assay (ELISA)). JIMT-1 (DSMZ,Braunschweig, Germany) was derived from the pleural effusion of a womanwith ductal breast cancer (grade 3 invasive, stage IIB) followingpostoperative radiation. JIMT-1 overexpresses HER2 at what is consideredto be a “medium” level of overexpression, but is insensitive toHER2-inhibiting drugs (e.g. trastuzumab). COLO 205 (ATCC) was derivedfrom the ascites fluid of man with carcinoma of the colon. COLO 205expresses carcinoembryonic antigen (CEA), keratin, interleukin 10(IL-10), and is considered to overexpress HER2 at relatively “low” levelof overexpression.

Tumor cells from each cell line were separately re-suspended in PBS with0.1% fetal bovine serum (FBS) at 1 to 10×10⁶ cells/mL. Cells weresubsequently incubated with 2 μM carboxyfluorescein succinimidyl ester(CFSE) to yield a final concentration of 1 μM. The reaction was quenchedafter 2 minutes via the addition of 10 mL complete medium with 10% FBSand washed twice with complete medium. Cells were either fixed in 2%paraformaldehyde and washed three times with PBS or left viable prior touse.

APC-Tumor Co-cultures. 2×10⁵ APCs were incubated with (e.g., FIG. 1A-1I)or without (e.g., FIG. 2A-3D) CFSE-labeled tumor cells between a 5:1 and10:1 effector to target (tumor) cell ratio in 96-well plates (Corning,Corning, N.Y.) containing iscove's modified dulbecco's medium (IMDM)(Thermo Fisher Scientific, Waltham, Mass.) supplemented with 10% FBS,100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, sodiumpyruvate, non-essential amino acids, and, where indicated, variousconcentrations of unconjugated HER2 antibody, Immunoconjugate A,Immunoconjugate B, and Immunoconjugate C of the invention (as preparedaccording to the examples above). Cells and cell-free supernatants wereanalyzed after 18 hours via flow cytometry or ELISA.

The results of this assay are shown in the figures, for example, FIG. 1A(CD40) and FIG. 1B (CD86) for Immunoconjugate A on the HCC1954 cellline, FIG. 1D (CD40) and FIG. 1E (CD86) for Immunoconjugate A on theJIMT-1 cell line, and FIG. 1G (CD40) and FIG. 1H (CD86) forImmunoconjugate A on the COLO 205 cell line.

FIG. 2A shows that Immunoconjugate B elicits myeloid differentiation asindicated by CD14 downregulation. FIG. 2B shows that Immunoconjugate Belicits myeloid activation as indicated by CD40 upregulation. FIG. 2Cshows that Immunoconjugate B elicits myeloid activation as indicated byCD86 upregulation. FIG. 3A shows that Immunoconjugate C elicits myeloiddifferentiation as indicated by CD14 downregulation. FIG. 3B shows thatImmunoconjugate C elicits myeloid activation as indicated by CD40upregulation. FIG. 3C shows that Immunoconjugate C elicits myeloidactivation as indicated by CD86 upregulation.

While the expression of T cell stimulatory molecules such as CD40 andCD86 are necessary for effective T cell activation, APCs also influencethe nature of the ensuing immune response through the secretion ofproinflammatory cytokines. Therefore, the capacity of immunoconjugatesto elicit cytokine secretion in human APCs following stimulation wasinvestigated. The data indicate that the immunoconjugate-stimulatedcells secreted high levels of TNFα. See FIG. 1C for Immunoconjugate Aco-cultured with the HCC1954 cell line, FIG. 1F for Immunoconjugate Aco-cultured with the JIMT-1 cell line, and FIG. 1I for Immunoconjugate Aco-cultured with the COLO 205 cell line. FIG. 2D shows TNFα secretionfrom myeloid cells following an 18 hour incubation with ImmunoconjugateB. FIG. 3D shows TNFα secretion from myeloid cells following an 18 hourincubation with Immunoconjugate C.

Example 20. Assessment of the Pharmacokinetics (PK) Properties ofImmunoconjugate B and Immunoconjugate C

This example shows that Immunoconjugate B and Immunoconjugate C havefavorable PK properties.

Cynomolgus primates (Macaca fascicularis) were dosed with 10 mg/kg ofImmunoconjugate B, Immunoconjugate C, Immunoconjugate D, ImmunoconjugateE, Immunoconjugate F, or Immunoconjugate G, as shown in Scheme 1, andthe PK properties were assessed for 28 days following administration.

A trastuzumab PK assay was configured to capture trastuzumab with HCA169anti-idiotype mAb and to detect with peroxidase labeled HCA176(HCA176P). An antibody drug conjugate assay was configured to capturetrastuzumab with HCA169 anti-idiotype mAb and to detect with a rabbitmAb to A103 followed by detection with peroxidase labeled Goatanti-rabbit IgG. Immunoconjugate B and Immunoconjugate C demonstratedhigher serum levels in both PK assays as compared to Immunoconjugate D,Immunoconjugate E, Immunoconjugate, F, and Immunoconjugate G.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An immunoconjugate of formula:

or pharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to 10, and “Ab” is an antibody construct comprising afirst variable region comprising: SEQ ID NO: 2 as light chaincomplementary determining region-1 (CDRL1); SEQ ID NO: 3 as light chaincomplementary determining region-2 (CDRL2); and SEQ ID NO: 4 as lightchain complementary determining region-3 (CDRL3), and a second variableregion comprising: SEQ ID NO: 5 as heavy chain complementary determiningregion-1 (CDRH1); SEQ ID NO: 6 as heavy chain complementary determiningregion-2 (CDRH2); and SEQ ID NO: 7 as heavy chain complementarydetermining region-3 (CDRH3).
 2. The immunoconjugate of claim 1, orpharmaceutically acceptable salt thereof, wherein subscript r is aninteger from 1 to
 6. 3. The immunoconjugate of claim 1, orpharmaceutically acceptable salt thereof, wherein subscript r is
 1. 4.The immunoconjugate of claim 1, or pharmaceutically acceptable saltthereof, wherein subscript r is
 2. 5. The immunoconjugate of claim 1, orpharmaceutically acceptable salt thereof, wherein subscript r is
 3. 6.The immunoconjugate of claim 1, or pharmaceutically acceptable saltthereof, wherein subscript r is
 4. 7. A composition comprising aplurality of immunoconjugates or pharmaceutically acceptable saltsthereof according to claim 1 and a pharmaceutically acceptable carrier.8. The composition of claim 7, wherein the composition further comprisesone or more pharmaceutically acceptable excipients.
 9. The compositionof claim 7, wherein

in the immunoconjugate is an adjuvant, and the composition has anaverage adjuvant to antibody ratio of from about 1 to about
 10. 10. Thecomposition of claim 9, wherein the composition has an average adjuvantto antibody ratio of from about 1 to about
 6. 11. The composition ofclaim 9, wherein the composition has an average adjuvant to antibodyratio of from about 1 to about
 4. 12. The composition of claim 9,wherein the composition has an average adjuvant to antibody ratio offrom about 1 to about
 3. 13. A method for treating cancer comprisingadministering a therapeutically effective amount of an immunoconjugateor pharmaceutically acceptable salt thereof according to claim 1 to asubject in need thereof.
 14. The method of claim 13, wherein the canceris a HER2-expressing cancer.
 15. The method of claim 13, wherein thecancer is breast cancer.
 16. The method of claim 13, wherein the breastcancer is HER2 overexpressing breast cancer.
 17. The method of claim 13,wherein the cancer is gastric cancer.
 18. The method of claim 13,wherein the gastric cancer is HER2 overexpressing gastric cancer. 19.The method of claim 13, wherein the cancer is gastroesophageal junctionadenocarcinoma.
 20. The method of claim 13, wherein the cancer isendometrial cancer.
 21. The method of claim 13, wherein the cancer isovarian cancer.
 22. The method of claim 13, wherein the cancer isuterine cancer.
 23. The method of claim 13, wherein the cancer isbladder cancer.
 24. The method of claim 13, wherein the cancer is lungcancer.
 25. The method of claim 13, wherein the cancer is head and neckcancer.
 26. The method of claim 13, wherein the cancer is liver cancer.27. The method of claim 13, wherein the cancer is colon cancer.
 28. Themethod of claim 13, wherein the cancer is melanoma.
 29. A method fortreating cancer comprising administering a therapeutically effectiveamount of a composition according to claim 7 to a subject in needthereof.
 30. The method of claim 29, wherein the cancer is aHER2-expressing cancer.
 31. The method of claim 29, wherein the canceris breast cancer.
 32. The method of claim 29, wherein the breast canceris HER2 overexpressing breast cancer.
 33. The method of claim 29,wherein the cancer is gastric cancer.
 34. The method of claim 29,wherein the gastric cancer is HER2 overexpressing gastric cancer. 35.The method of claim 29, wherein the cancer is gastroesophageal junctionadenocarcinoma.
 36. The method of claim 29, wherein the cancer isendometrial cancer.
 37. The method of claim 29, wherein the cancer isovarian cancer.
 38. The method of claim 29, wherein the cancer isuterine cancer.
 39. The method of claim 29, wherein the cancer isbladder cancer.
 40. The method of claim 29, wherein the cancer is lungcancer.
 41. The method of claim 29, wherein the cancer is head and neckcancer.
 42. The method of claim 29, wherein the cancer is liver cancer.43. The method of claim 29, wherein the cancer is colon cancer.
 44. Themethod of claim 29, wherein the cancer is melanoma.